Oxy-vanadium (iv) complexes having spermicidal activity

ABSTRACT

Organometallic oxovanadium (IV) complexes having potent spermicidal activity, particularly those having at least one bidentate ligand. Preferred compounds are stable oxovanadium (IV) complexes having, as ancillary ligands linked via nitrogen or oxygen-metal bonds, bis and/or mono-1,10-phenanthroline; 2,2′-bipyridyl; or 2-hydroxyacetophenone.

This application is a divisional of application Ser. No. 09/187,115,filed Nov. 5, 1998, U.S. Pat. No. 6,245,808, which application(s) areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compositions containing oxy-vanadium (IV).More particularly, the invention relates to oxy-vanadium (IV) containingcomplexes having spermicidal activity.

BACKGROUND OF THE INVENTION

The known spermicidal agents, nonoxynol-9 and gramicidin, exert theireffects via a detergent-like ability to damage the sperm plasmamembrane, perturb its conformation and destroy its semi-permeable naturethereby impairing the sperm motility and egg fertilizing functions(Wilborn, et al., Fertil Steril 1983; 39:717-719; Bourinbaiar, et al.,Life Sci 1994; 54:PL 5-9). Because of their non-specific membranedisruptive properties, such vaginal spermicides have been shown todamage the cervicovaginal epithelium, as well, which may lead to a lowerdegree of protection from sexually transmitted diseases (Niruthisard, etal., Sex Transm Dis 1991; 18:176-179). A novel vaginal contraceptivepreferably does not function with the non-specific membrane toxicitymediated by detergent-type action of the currently available vaginalcontraceptives.

Vanadium is a physiologically essential element which can be found inone to five (I to V) oxidation states. Several inorganic saltscontaining vanadium with oxidation state +4 (IV) have been shown tofunction as modulators of cellular redox potential and to exertpleiotropic effects in multiple biological systems by catalyzing thegeneration of reactive oxygen intermediates. See, for example, Shi, etal., Ann Clin Lab Sci 1996; 26:390-49; Byczkowski, et al., Bull EnvironContam Toxicol 1988; 41:696-703; Younes, et al., Toxicology 1991;66:63-74, and Sakurai, et al., Biochem Biophys Res Commun 1995;206:133-137. Reactive oxygen intermediates have been reported to affectsperm motility by a combination of peroxidation of membrane lipids andproteins (Aitken, et al., Biol Reprod 1989; 40:183-197; Jones, et al.,Fertil Steril 1979; 31:531-537). Peroxidative damage to the sperm plasmamembrane is an important pathophysiological mechanism in the onset ofmale infertility (Aitken, et al., BioEssays 1994; 16:259-267). It hasalso been shown that superoxide radicals generated by the action ofxanthine oxidase exert a direct, suppressive effect on many aspects ofsperm function (Aitken, et al., J. Reprod. Fertil. 1993; 97:441-450).Sperm are thought to be particularly susceptible to oxidative stress byvirtue of their high content of unsaturated fatty acids and theirrelative paucity of cytoplasmic enzymes for scavenging the reactiveoxygen intermediates that initiate lipid peroxidation (Alvarez, et al.,J Androl 1987; 8:338-348).

There is a need for new spermicidal compounds for contraceptivepurposes. The ability of vanadium (IV) containing organometalliccomplexes to catalyze the generation of reactive oxygen species and thespermicidal activity of these agents was described in copending patentapplication U.S. Ser. No. 09/008,898, which is hereby incorporated byreference for all purposes.

Metallocene complexes containing vanadium (IV) as the central metal ionwithin the tetrahedral bis(cyclopentadienyl) [Cp₂] metal complex(vanadocene) have potent spermicidal activity against human sperm (BiolReprod 58:1516,1998; Molec Hum Reprod 4:683, 1998). The spermicidalactivity was dependent on vanadium (IV) as the central metal ion withinthe Cp₂-metal complex, but the various diacido groups and bidentateligands coordinated to the Cp₂-vanadium (IV) moiety also significantlymodulated the spermicidal potency.

SUMMARY OF THE INVENTION

Organometallic complexes containing oxovanadium (IV) (VO) have now beenfound to have potent, concentration-dependent spermicidal activity atmicromolar concentrations. Preferred oxovanadium complexes of theinvention are complexes having at least one bidentate ligand. Suitablebidentate ligands include N,N′; N,O; and O,O′ bidentate ligands.

The spermicidal activity of oxovanadium (IV) complexes was found to beirreversible, since the treated sperm underwent apoptosis, as determinedby the flow cytometric quantitation of mitochondrial membrane potential,surface Annexin V binding assay, and in situ DNA nick-end labeling ofsperm nuclei. The percentages of apoptotic sperm quantitated by theseflow cytometric assays correlated well with the spermicidal potency ofoxovanadium (IV) complexes. These results provide unprecedented evidencethat the spermicidal and apoptosis-inducing properties of diverseoxovanadium (IV) complexes is due to vanadium (IV) as the central metalion within the oxovanadium (IV) complex. The oxovanadium (IV) complexestypically included at least one bidentate ligand within the complex.Suitable bidentate ligands include N,N′; N,O; and O,O′ bidentateligands. Examples of suitable bidentate ligands include bipyridyl,bridged bipyridyl and acetophenone ligands modulating the spermicidalpotency. One example of a bridged bipyridyl includes phenanthroline.These novel oxovanadium (IV) complexes, and particularly thebromo-hydroxyacetophenone complex [OV(Br,OH-acph)₂], are useful ascontraceptive agents.

Accordingly, the present invention includes spermicidal oxovanadiumcomplexes, as well as contraceptive compositions containing aspermicidal effective amount of an oxovanadium IV complex and apharmaceutically acceptable carrier, diluent or vehicle. The spermicidalcompounds of the invention include organometallic oxovanadium (IV)complexes. Preferably, the oxovanadium (IV) is complexed with at leastone bidentate ligand.

One suitable embodiment of the invention having a bidentate ligandwherein the bidenate ligand is a bipyridyl has the general formula I,shown below:

where R and R¹ are the same or different and are independently selectedfrom: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano,carboalkoxy (e.g. C₂-C₆) and nitro; X and X¹ are the same or differentand are independently selected from: monodenate and bidentate ligands;and n is 0 or 1.

Another suitable embodiment of the invention having a bidentate ligandwherein the bidentate ligand is a bridged bipyridyl has the generalformulae IV, shown below:

where R² and R³ are the same or different and are selected from H, loweralkyl, halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy(e.g. C₂-C₆) and nitro; X² and X³, are the same or different and areselected from monodentate and bidentate ligands; Z is selected from O,CH₂, CH₂—CH₂, and CH═CH; and n is 0 or 1.

Another suitable embodiment of the invention having a bidentate ligandwherein the bidentate ligand is a bridged bipyridyl, and the bridgedbipyridyl is phenanthroline, has the general formula II, shown below:

where R⁴, R⁵ and R⁶ are the same or different and are independentlyselected from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl,cyano, carboalkoxy (e.g. C₂-C₆) and nitro; X⁴ and X⁵ are the same ordifferent and independently selected from: monodentate and bidentateligands; and n is 0 or 1.

Another suitable embodiment of the invention having a bidentate ligandwherein the bidentate ligand is an O,O′ bidentate ligand, and thecomplex has the general formulae III, is shown below:

where R⁷ and R⁹ are the same or different and are independently selectedfrom: H, lower alkyl, lower alkoxy, and halogenated alkyl; R⁸ isselected from H, lower alkyl, halo, lower alkoxy, and halogenated alkyl;Y and Y¹ are the same or different and independently selected from thegroup consisting of: monodentate and bidentate ligands; and n is 0 to 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural drawing of a representative compounds V-XV of theinvention.

FIG. 2 is a graph showing spermicidal activity (loss of sperm mobility)after treatment with the spermicidal oxovanadium (IV) complexes of theinvention.

FIGS. 3A and 3B are bar graphs showing the inhibition of sperm motionparameters measured by CASA after treatment with VO (Cl-phen)₂.

FIGS. 4A-4F are scans demonstrating VO-complex induced apoptosis intreated sperm analyzed by flow cytomeric quantitation.

FIGS. 5A-5C are confocal laser scanning microscopy images of spermnucleic undergoing apoptosis following treatment with VO(Cl-phen)₂.

DETAILED DESCRIPTION

As used herein, the following definitions define the stated terms:

“Organometallic compound” is an organic compound comprised of a metalattached directly to carbon (R—M).

“Coordination compound” is a compound formed by the union of a centralmetal atom or ion with a nonmetal atom, ion or molecule called a ligandor complexing agent.

“Ligand” or a “complexing agent” is a molecule, ion or atom that isattached to the central metal atom or ion of a coordination compound.

“Monodentate ligand” is a ligand having a single donor atom coordinatedto the central metal atom or ion.

“Bidentate ligand” is a ligand having two donor atoms coordinated to thesame central metal atom or ion.

“Oxovanadium (IV) complex” is a coordination compound including vanadiumas the central metal atom or ion, and the vanadium has an oxidationstate of +4 (IV), and is double bonded oxygen.

The present invention concerns or organometallic oxovanadium complexes,and the finding that such oxovanadium complexes have potent andselective spermicidal activity, and are particularly active and stablespermicidal agents.

Another aspect of the present invention is a method of contraceptionincluding the step of contacting sperm with a spermicidal effectiveamount of an oxovanadium IV complex.

Vanadium

Vanadium is a physiologically essential element that can be found inboth anionic and cationic forms with oxidation states ranging from −3 to+5 (I-V). This versatility provides unique properties to vanadiumcomplexes. In particular, the cationic form of vanadium complexes withoxidation state +4 (IV) have been shown to function as modulators ofcellular redox potential, regulate enzymatic phosphorylation, and exertpleiotropic effects in multiple biological systems by catalyzing thegeneration of reactive oxygen species (ROS). Besides the ability ofvanadium metal to assume various oxidation states, its coordinationchemistry also plays a key role in its interactions with variousbiomolecules. In particular, organometallic complexes of vanadium (IV)linked to bis(cycopentadienyl) moieties or vanadocenes exhibit antitumorproperties both in vitro and in vivo primarily via oxidative damage.

Human Sperm

Human sperm are known to be exquisitely sensitive to oxidative stressdue to the high content of polyunsaturated fatty acids in their cellmembranes, the low levels of cytoplasmic enzymes (superoxide dismutase,catalase, and gluthathione peroxidase) for scavenging the reactionoxygen species (ROS) intermediates, the initiate lipid peroxidation, andreduced activity of repair enzymes (exonucleases, endonucleases,glycosidases, and polymerases) to recover from oxidative damage.Reactive oxygen species such as a hydrogen peroxide (H₂O₂) and hydroxylradicals (*OH) have been shown to affect sperm motility by a combinationof peroxidation of membrane lipids and proteins. Oxidative damage tosperm proteins, carbohydrates, and DNA is an importantpathophysiological mechanism in the onset of male infertility. It hasalso been shown that superoxide radicals generated by the action ofxanthine oxidse exert a direct, suppressive effect on sperm function,including loss of motility, impaired capacitation, and sperm-egginteraction.

Our recent studies of 12 monodentate and 7 bidentatebis(cyclopentadienyl)vanadium (IV) complexes showed that thesevanadocenes have potent spermicidal and apoptosis-inducing propertiesagainst human sperm. In fact, very short (<1 minute) exposure tovanadocenes at nanomolar to micromolar concentrations was sufficient toinduce complete sperm motility less, whereas, prolonged exposure ofsperm to millimolar concentrations of inorganic vanadium (oxidationstate IV and V) salts had no effect on sperm motility (see copendingpatent application U.S. Ser. No. 09/008,898). Furthermore, none of theother metallocenes (oxidation state IV) containing titanium, zirconium,molybdenum, or hafnium exhibited spermicidal activity.

Since the redox potential and the stability of organovanadium complexesare greatly affected by the complexed ligands, different ligands wereselected to test their effects on the spermicidal activity andstability. Diverse complexes were synthesized, containing mono andbidentate ligands complexed with the central vanadium (IV) by carbon-,nitrogen-, or oxygen- metal bonds.

The stability of organometallic complexes with monodentate ligands inaqueous solutions was found to be improved by chelating effects ofcertain bidentate ligands, particularly dithiocarbamate andacetylacetonate (D'Cruz et al., 1998, Mol. Hum. Reprod. 4:683-693).Stable oxovanadium (IV) complexes were synthesized having, as ancillaryligands linked via nitrogen or oxygen-metal bonds, bis and/ormono-1,10-phenanthroline (phen); 2,2′- bipyridyl (bipy); or2-hydroxyacetophenone (acph). Specific compounds included the following:

phen bipy acph VO(phen) VO(bipy) VO(Br,OH-acph)₂ VO(phen)₂ VO(bipy)₂VO(Me₂-phen) VO(Me₂-bipy) VO(Me₂-phen)₂ VO(Me₂-bipy)₂ VO(Cl-phen)VO(Cl-phen)₂

The phenanthroline complex of peroxivanadate potentiates the generationof ROS in cells (Sakurai et al., 1995, BBRC 206:133-137). Thesecompounds were synthesized and examined for potential spermicidalactivity using computer-assisted sperm analysis. (CASA). Resultspresented below provide unprecedented evidence that oxo-vanadiumcomplexes, including oxovanadium IV-bound to phenanthroline, bipyridyland 2-hydroxyacetophenone as ancillary ligands and their derivatives arepotent spermicidal agents and also induce apoptosis in human sperm.

Compounds

Compounds of the invention include oxovanadium (IV) containingorganometallic complexes having spermicidal activity. Preferred theoxovanadium (IV) complexes include at least one bidentate ligand.Suitable bidentate ligands include N,N′; N,O: and O,O′ bidentateligands. Examples of suitable bidentate ligands include bipyridyl,bridged bipyridyl, and acetophenone. Particularly, preferred oxovanadiumcompounds of the invention are those having the formulas I, II, III, andIV shown and described below.

A suitable embodiment of the invention includes a bidentate ligandwherein the bidentate ligand is a bipyridyl and the oxovanadium IVcomplex has the general formulae I, below:

where R and R¹ are the same or different and are independently selectedfrom: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano,carboalkoxy (e.g. C₂-C₆) and nitro; X and X¹ are the same or differentand are independently selected from: monodentate and bidentate ligands;and n is 0 or 1.

Another suitable embodiment of the invention has a bidentate ligandwherein the bidentate ligand is a bridged bipyridyl and the oxovanadiumIV complex has the general formulae IV, below:

where R² and R³ are the same or different and are selected from H, loweralkyl, halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy(e.g. C₂-C₆) and nitro; X² and X³, are the same or different and areselected from monodentate and bidentate ligands; Z is selected from O,CH₂, CH₂—CH₂, and CH═CH; and n is 0 is 1.

Another suitable embodiment of the invention has a bidentate ligandwherein the bidentate ligand is a bridged bipyridyl, and the bridgedbipyridyl is phenanthroline, and the oxovanadium IV complex has thegeneral formulae II, below:

where R⁴, R⁵ and R⁶ are the same or different and are independentlyselected from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl,cyano, carboalkoxy (e.g. C₂-C₆) and nitro; X⁴ and X⁵ are the same ordifferent and independently selected from: monodentate and bidentateligands; and n is 0 or 1.

Another suitable embodiment of the invention has a bidentate ligandwherein the bidentate ligand is an O,O′ bidentate ligand, theoxovanadium IV complex has the general formulae III, below:

where R⁷, and R⁹ are the same or different and are independentlyselected from: H, lower alkyl, lower alkoxy, and halogenated alkyl; R⁸is selected from H, lower alkyl, halo, lower alkoxy, and halogenatedalkyl; Y and Y¹ are the same or different and independently selectedfrom the group consisting of: monodentate and bidentate ligands; and nis 0 or 1.

Preferred monodentate ligands for the oxovanadium complex include H₂O,halides and carboxylates. Preferred bidentate ligands include N,N′bidentate ligands, N, O bidentate ligands, and O, O′ bidentate ligands.Examples of suitable N, N′ bidentate ligands include diamines and othersuch known suitable N, N′ bidentate ligands. Examples of diaminesinclude bipyridal, derivatives of bipyridal, bridged bipyridal, such asphenanthroline, derivatives of phenanthroline, and other such compounds.Examples of suitable N, O bidentate ligands include amino acids andSchiff base type groups. Examples of suitable O, O′ bidentate ligandsinclude dicarboxylate, 2-hydroxyacetophenone, acetylacetone type andcatechol type groups.

Particularly useful compounds of the invention are those shown in FIG.1, compounds V-XV.

Spermicidal Use in Mammals

The spermicidal compositions of the present invention are suitable foruse in mammals. As used herein, the term “mammals” means any class ofhigher vertebrates that nourish their young with milk secreted bymammary glands, e.g., humans, rabbits and monkeys.

The spermicides useful in accordance with the present invention includethe above-mentioned oxovanadium complexes where the vanadium metal hasan oxidation state of +4.

Thus, the contraceptive compositions of the present invention containone of the above-mentioned organometallic oxovanadium complexes. Thetotal amount of spermicide thereof will typically range from about 0.05to 0.5 weight percent based on the weight of the contraceptivecomposition. Preferably, the amount of spermicide employed will be thatamount necessary to achieve the desired spermicidal results. Appropriateamounts can be determined by those skilled in the art. Preferably, theamount of the spermicide employed, a spermicidal effective amount, willcomprise from about 0.0025 to 0.025 weight percent, and more preferablyfrom about 0.05 to 0.5 weight percent, based on the weight of thecontraceptive composition.

When used in vivo to selectively kill testicular germ cells oftesticular germ cell tumors, the administered dose is that effective tohave the desired effect, e.g., sufficient to kill essentially all normalgerm cells for chemical castration, or sufficient to reduce or eliminatea testicular cell tumor. The appropriate dose can be extrapolated usingknown methods and relationships. A useful dose will vary with thedesired effect, the mode of administration, and the compositionadministered. In general, the desired dose will be in the range of 1-25mg/kg body weight.

The compositions of the invention contain not only the spermicide butnecessarily pharmaceutically acceptable carriers, diluents or vehicles,i.e., one that appropriately delivers oxovanadocene complexes to a sitefor contact with sperm or germ cells and provides spermicidal and/oranti-germ cell activity.

One advantageous component in the pharmaceutical composition foradministration of a spermicide is a polymeric delivery component asdescribed in U.S. Pat. No. 5,595,980, which patent is incorporatedherein by reference. It has been found that such polymeric deliverycomponent enhances effectiveness of a spermicide and reduces vaginalirritation on administration.

In addition to the polymeric component, the balance of the contraceptivecompositions, i.e., typically from about 0.1 to 99.8% and often about 50to 99.8 weight percent, may optionally comprise one or more cosmeticingredients. Such cosmetic ingredients are known to those skilled in theart and are often referred to in the art as diluents, solvents andadjuvants. Typically cosmetic ingredients include, for example; water,ethyl alcohol, isopropyl alcohol, glycerin, glycerol propylene glycol,sorbitol and other high molecular weight alcohols. In addition,contraceptive compositions may contain minor amounts, e.g. from about0.1 to 5% weight based on the weight of the contraceptive compositions,of other additives, such as, for example; stabilizers, surfactants,menthol, eucalyptus oil, other essential oils, fragrances, and the like.Polyoxyethylene 20 sorbitan monolaurate is a preferred stabilizer foruse in the compositions. Details concerning the selection and amounts ofcosmetic ingredients, other additives, and blending procedures are knownto those skilled in the art.

The contraceptive compositions of the present invention may be deliveredto the vagina of a mammal by any means known to those skilled in theart. Typical forms for delivery of the compositions include, forexample; creams, lotions, gels, foams, sponges, suppositories and films.In addition, the compositions of the present invention may be used aspersonal care lubricants, such as, for example, condom lubricants, andthe like. Such lubricants may comprise commonly known ingredients suchas, for example: humectants; e.g., glycerin, sorbitol, mannitol, glycolsand glycol ethers; buffers, e.g., glucono-d-lactone; germicides orbactericides; e.g., chlorhexidine gluconate; preservatives, e.g.,methylparaben; viscosifiers; e.g., hydroxyethyl cellulose, etc.; otheradjuvants; e.g., colors and fragrances; in addition to the compositionsof the present invention. Those skilled in the art will recognize thatthe physical properties, e.g., viscosity, of such delivery forms mayvary widely. For example, the viscosity of gel form of the compositionof the present invention, e.g., 150,000 centipoise, may be substantiallyhigher than the viscosity of lotion form of the composition of thepresent invention e.g., 100 centipoise. The contraceptive compositionsmay be located within a condom for example. Further details concerningthe materials, ingredients, proportions and procedures of such deliveryforms are known to those skilled in the art.

The contraceptive compositions of the present invention are preferablyadministered to the vagina of the mammal in a dosage which is effectiveto immobilize sperm present in the vagina and/or to inhibit theirpenetration in cervical mucus. Typical dosages range between about0.0001 to 0.001 grams of the compositions per kilogram of body weight ofthe mammal.

Inter-vaginal devices may also be used to aid in the administration ofthe spermicide as described in U.S. Pat. No. 5,069,906.

In administration the spermicide in the form of the above compositions,the compositions may also be formulated to release the spermicide bothrapidly and with a prolonged release of the drug. Such a formationproviding both rapid and prolonged release has been described in U.S.Pat. No. 4,707,362, which patent is also incorported herein.

In administering the spermicide in vivo, it is understood that multipledelivery methods are available, including injection both systemic andlocal. The preferred method of delivery is local, e.g., intratesticularinjection. Where appropriate, the composition may be directly injectedinto a testicular germ cell tumor mass.

The invention may be further clarified by reference to the followingExamples, which serve to exemplify some of the preferred embodiments,and not to limit the invention.

EXAMPLES Example 1 Oxovanadium (IV) Complexes Containing1,10-Phenanthroline, 2,2′-Bipyridyl or 5′-Bromo-2′-Hydroxyacetophenoneand Derivatives

Materials and Methods

The chemical structures of the various mono and bis 1,10-phenanthroline,2,2′-bipyridyl and 2-hydroxyacetophenone complexes of oxovanadium (IV)synthesized and analyzed in this study are depicted in FIG. 1. Thecationic complexes synthesized and tested with 1,10-phenanthroline(phen), 2,2′-bipyridyl (bipy) and their derivatives as ancillary ligandswith general formulas of [VO(L)(H₂O)₂](SO₄) and [VO(L)₂(H₂O)](SO₄)(L=ligand) are:

(1) [(VO(phen)]=(diaqua)(1,10-phenanthroline)oxovanadium (IV) sulfate;

(2) [VO(phen)₂]=(aqua)bis(1,10-phenanthroline)oxovanadium (IV) sulfate;

(3) [VO(Me₂-phen)]=(diaqua)(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate;

(4)[VO(Me₂-phen)₂]=(aqua)bis(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate;

(5) [VO(Cl-phen)]=(diaqua)(5-chloro-1,10-phenanthroline)oxovanadium (IV)sulfate;

(6) [VO(Cl-phen)₂]=(aqua)bis(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate;

(7) [VO(bipy)]=(diaqua)(2,2′-bipyridyl)oxovanadium (IV) sulfate;

(8) [VO(bipy)₂]=(aqua)bis(2,2′-bipyridyl)oxovanadium (IV) sulfate;

(9) [VO(Me₂-bipy)]=(diaqua)(4,4′-dimethyl-2,2′-bipyridyl)oxovanadium(IV) sulfate;

(10) [VO(Me₂-bipy)₂]=(aqua)bis(4,4′-dimethyl-2,2′-bipyridyl)oxovanadium(IV) sulfate;

(11) [VO(Br,OH-acph)₂]=bis(5′-bromo-2′-hydroxyacetophenone)oxovanadium(IV)); and

(12) 5′-bromo-2′-hydroxyacetophenone was synthesized as a neutralcomplex.

Chemical Synthesis:

The 9 novel oxovanadium (IV) complexes were synthesized based onpreviously published chemistry of VO(phen) and VO(phen)₂ complexes(Sakurai et al., 1995, BBRC 206:133-137). Briefly, these complexes weresynthesized by reacting an aqueous solution of vanadyl sulfate with anethanol solution of a chloroform solution of the ligands. The complexespurified from chloroform, ether and/or water were characterized byFourier transform infrared spectroscopy (FT-Nicolet model Protege 460;Nicolet Instrument Corp., Madison, Wis.), UV-visible spectroscopy (DU7400 spectophotometer; Beckman Instruments, Fullerton, Calif.), massspectrometry (Finnigan MAT 95 mass spectrometer, Madison, Wis.) andelemental analysis (Atlantic Microlab, Inc., Norcross, Ga.). Theseoxovanadium (IV) complexes have a square pyramidal geometry with the oxoligand (O²⁻) in the apical site. The peroxivanadates are stabilized withbidentate ligands which form a 5-membered ring with the vanadium atom.The choice of these three organic ligands (phenanthroline, bipyridyl,and acetophenone) was based on the reported fact that the cationicoxovanadium (IV) complex of phenanthroline is superior to cisplatin(cis-diamminedichloroplatinum[II]) with respect to antitumor activity(Sakurai et al., 1995, Supra), the structural similarity of bipyridylring to phenanthroline, as well as the neutral nature of acetophenonecomplex of oxovanadium (IV). Structural variations of the ligandsincluded addition of bromo, chloro or methyl groups on thephenanthroline, bipyridyl or acetophenone rings.

Sperm Immobilization Assay (SIA)

To evaluate the spermicidal effects of complexes of oxovanadium (IV),VO(phen), VO(phen)₂, VO(Me₂—phen), VO(Me₂—phen)₂, VO(Cl—phen),VO(Cl—phen)₂], VO(bipy), VO(bipy)₂, VO(Me₂—bipy), VO(Me₂—bipy)₂, andVO(Br,OH—acph)₂, a highly motile fraction of pooled donor sperm (N=5)was prepared by discontinuous (90−45%). Percoll gradient (ConceptionTechnologies, San Diego, Calif.) centrifugation and the “swim-up” methodas described previously (Aitken et al., 1989, Biol. Reprod. 40:183-197).All donor specimens were obtained after informed consent and incompliance with the guidelines of the Hughes Institutional Review Board.Motile sperm (≳10×10⁶/ml) were suspended in 1 ml of Biggers, Whitten,and Whittingham's medium (BWW) containing 0.3% BSA (fraction V; SigmaChemical Co., St. Louis, Mo.) in the presence and absence of serial2-fold dilutions of test substance (250 μM−1.9 μM) in 0.25% dimethylsulfoxide (DMSO). For each experiment, fresh stock solutions (100 mM) ofvanadium compounds were prepared in DMSO. A corresponding volume of DMSO(0.25%) was added to control sperm suspension. After 3 hours ofincubation at 37° C., the percentage of motile sperm was evaluated bycomputer-assisted sperm motion analysis (CASA) as described previously(Aitken et al., 1989, Supra). The percentages of motilities werecompared with those of sham-treated control suspensions of motile sperm.The spermicidal activity of test compounds was expressed as the EC₅₀values (the final concentration of the compound in medium that decreasesthe proportion of motile sperm by 50%).

To test the effect of duration of incubation on sperm immobilization inthe presence of oxovanadium (IV) complexes, a motile fraction of sperm10⁷/ml) was incubated at 37° C. in 1 ml of BWW−0.3% BSA in the presenceof 200 μM each of the complexes or 0.2% DMSO alone. At timed intervals(every 5 and 10 minutes) aliquots (4 μl) were transferred to two 20 μmMicrocell (Conception Technologies) chambers, and sperm motility wasassessed by CASA.

Sperm Kinematic Parameters

For CASA, 4 μl each of sperm suspension was loaded into two 20 μmMicrocell chambers placed onto a counting chamber at 37° C. At least 5-8fields per chamber were scanned for analysis using a Hamilton ThorneIntegrated Visual Optical System (IVOS) version 10 instrument (HamiltonThorne Research Inc., Danvers, Mass.). Each field was recorded for 30seconds. The Hamilton Thorne computer calibrations were set at 30 framesat a frame rate of 30 images/seconds. Other settings were as follows:minimum contrast 8; minimum size 6; low-size gate, 1.0; high-sizegate,2.9; low-intensity gate, 0.6; high-intensity gate, 1.4;phase-contrast illumination; low path velocity at 10 μm/seconds andthreshold straightness at 80%; magnification factor, 1.95. Theperformance of the analyzer was periodically checked using the playbackfunction.

The attributes of sperm kinematic parameters evaluated included numberof motile (MOT) and progressively (PRG) motile sperm; curvilinearvelocity (VCL; a measure of the total distance traveled by a given spermduring the acquisition divided by the time elapsed); average pathvelocity (VAP; the spatially averaged path that eliminates the wobble ofthe sperm head), straight-line velocity (VSL; the straight-line distancefrom beginning to end of track divided by time taken), beat-crossfrequency (BCF; frequency of lateral head displacement), (ALH; the meanwidth of sperm head oscillation), and the derivatives, straightness(STR=VSL divided by VAP×100), linearity (LIN=VSL divided by VCL×100,departure of sperm track from a straight line). Data from eachindividual cell track were recorded and analyzed. At least 200 spermwere analyzed for each aliquot sampled.

Flow Cytometric Quantitation of Sperm Acrosome Reaction

In experiments designed to assess the comparative effects of oxovanadium(IV) complexes and N-9 on sperm acrosome reaction, motile fractions ofsperm (10⁷/ml) prepared from a single donor were incubated in 1 ml ofBWW−0.3% BSA in the presence of 100 μM each of the vanadocene complexes,VO(phen), VO(phen)₂, VO(Me₂—phen), VO(Me₂—phen)₂, VO(Cl—phen),VO(Cl—phen)₂, VO(bipy), VO(bipy)₂, VO(Me₂—bipy), VO(Me₂—bipy)₂, andVO(Br,OH—acph)₂ in 0.1% DMSO, N-9, or DMSO (0.1%) alone at 37° C. After3 hours, 5 μg/ml of purified, phycoerythrin (PE)-conjugated murineanti-CD46 monoclonal antibody (mAb; clone 122-2; Research Diagnostics,Flanders, N.J.) was added and the sperm suspensions incubated for anadditional 30 minutes. The suspensions was washed in Tyrode's saltsolution (Sigma) containing 1% BSA (1% TBSA) and the percentage ofCD46-positive sperm were analyzed by flow cytometry using a FACS Vantageflow cytometer (Becton Dickinson, Mountain View, Calif.), as describedpreviously (DeCruz et al., 1996, Biol Reprod. 54:1217-1228; DeCruz etal., 1992, Fertil. Seril., 58:633-636). Two separate experiments wereperformed to quantitate acrosomal loss following exposure of sperm tooxovanadium (IV) complexes.

Flow Cytometric Assays for Oxovanadium (IV) Complex-Induced Apoptosis

Three independent flow cytometric apoptotic assays were used todetermine oxovanadium (IV)-mediated quantitative changes at themitochondrial, surface membrane, and sperm nuclear compartments. Thesedata are shown in FIGS. 4A-4F.

Assessment of Mitochondrial Transmembrane Potential (ΔΨm) Using JC-1Dye:

The loss of ΔΨm, a early marker for apoptosis was quantitated by flowcytometry using the lipophilic cationic dye, 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolecarbocyanine iodide (JC-1) as describedin Cossarizza et al., 1993, BBRC 197:40-45. This dye accumulates in themitochondrial matrix under the influence of the ΔΨm. The molecule isable to selectively enter into mitochondria, the monomeric form emittingat 527 nm after excitation at 490 nm. However, depending on the membranepotential, JC-1is able to form J-aggregates that are associated with alarge shift in emission (590 nm). The color of the dye changesreversibly from green to greenish orange at ΔΨm becomes more polarized.

To quantitate changes in sperm ΔΨm following oxovanadium (IV) complexexposure, highly motile fraction of sperm (10⁷/ml) in duplicatealiquots, were incubated at 37° C. for 3 hours in BWW−0.3% BSA medium inthe presence and absence of 100 μM each of the vanadocene complexes,VO(phen), VO(phen)₂, VO(Me₂—phen), VO(Me₂—phen)₂, VO(Cl—phen),VO(Cl—phen)₂, VO(bipy), VO(bipy)₂, VO(Me₂—bipy), VO(Me₂—bipy)₂, andVO(Br,OH—acph)₂. Following incubation, 10 μg/ml JC-1 (Molecular Probes,Eugene, Oreg.) was added from a stock solution in DMSO (1 mg/ml) to thesperm suspension and incubated for an additional 10 minutes. At the endof the incubation period, sperm were washed in Tyrode's salt solution(Sigma), resuspended in 200 μl, and analyzed by flow cytometry forJC-1-specific fluorescence. The excitation was at 488 nm; the emissionsfor green and red/orange fluorescence were 530 nm and 575 nmrespectively. JC-1 monomer and aggregated fluorescence weresimultaneously measured in oxovanadium (IV) complex-exposed and controlsperm. The percentages of sperm positive for green, orange, and greenishorange were determined using the cutoff signals for JC-1 labeled motilesperm.

Assessment of Sperm Membrane Changes Using FITC Annexin V

In order to examine the expression of phosphatidyl serine on the spermsurface following oxovanadium (IV) complex exposure, surface binding ofFITC-Annexin V was evaluated by flow cyotometry as described in Vermeset al., 1995, J. Immunol. Meth., 1984:39-51. One ml aliquots of highlymotile sperm (10⁷) in triplicate were incubated in BWW−0.3% BSA at 37°C. for 12 hours with and without 100 μM of each of the oxovanadium (IV)complexes VO(phen), VO(phen)₂, VO(Me₂—phen), VO(Me₂—phen)₂, VO(Cl—phen),VO(Cl—phen)₂, VO(bipy), VO(bipy)₂, VO(Me₂—bipy), VO(Me₂—bipy)₂, andVO(Br,OH—acph)₂ in 0.1% DMSO. After exposure to these complexes, spermwere washed with 1% Tyrode's salt solution containing 1% BSA (1% TBSA),and the pellets were resuspended in the same medium. The spermsuspension was reacted for 30 minutes at room temperature with 6 μg/mlof FITC-conjugated recombinant human Annexin V (Caltag Laboratories, SanFrancisco, Calif.). After two washes in Tyrode's salt solution, spermwere resuspended in 1% TBSA containing 1 μg/ml propidium iodide (PI) andanalyzed for surface-bound Annexin V and PI-permeability by quantitativeflow cytometry using an argon laser for excitation of fluorescence.Annexin V and PI binding were simultaneously measured in oxovanadium(IV) complex-exposed and control sperm as described previously (deLamirande et al., 1993, Fertil Seril 59:1291-1295). The percentages ofsperm positive for Annexin V and PI were determined using the cutoffsignals for membrane-intact motile sperm. Two separate experiments wereperformed to assess the surface expression of phosphatidyl serinefollowing exposure of sperm to oxovanadium (IV) complexes.

Assessment of DNA-Fragmentation Using In Situ DNA Nick-End Labeling byTUNEL Method

A flow cytometric two-color terminal deoxynucleotidyl transferase (TdT)assay was employed to detect apoptotic sperm nuclei by TdT-mediated dUTPnick-end labeling (TUNEL, as described in Gavrieli et al., 1992, J. CellBiol., 119:493-501). The comparative effect of the oxovanadium (IV)complexes, VO(phen), VO(phen)₂, VO(Me₂—phen), VO(Me₂—phen)₂,VO(Cl—phen), VO(Cl—phen)₂, VO(bipy), VO(bipy)₂, VO(Me₂—bipy),VO(Me₂—bipy)₂, and VO(Br,OH—acph)₂, to induce apoptosis was tested byincubating 1 ml duplicate aliquots of motile sperm (10⁷/ml) in BWW−0.3%BSA at 37° C. for 24 hours with and without 100 μM each of the testcompounds. Sperm were washed in phosphate-buffered saline (PBS)−1% BSA,fixed in 4% paraformaldehyde in PBS for 15 minutes. Following twowashings in PBS, they were permeabilized with 0.1% Triton X-100 in 0.1%sodium citrate for 2 minutes on ice, and washed twice with PBS. New3′-hydroxyl (3′—OH) end labeling of fragmented sperm nuclear DNA wasperformed using TdT and detected by fluorescein (FITC)-conjugateduridine triphosphate (dUTP) according to the manufacturer'srecommendations (Boehringer-Mannheim, Indianapolis, Ind.). Spermaliquots incubated without TdT enzyme served as a negative control.Nonapoptotic sperm do not incorporate significant amounts of dUTP due tolack of exposed 3′—OH ends, and consequently have much less fluorescencecompared to apoptotic cells which have an abundance of 3′—OH ends.Oxovanadium (IV)-induced apopotosis of sperm was shown by an increase inthe number of cells staining with FITC- dUTP (M2 gates). The M1 and M2gates were used to demarcate non-apoptotic and apoptoticPI-counterstained sperm populations, respectively. Two separateexperiments were performed to assess dUTP incorporation followingexposure of sperm to oxovanadium (IV) complexes.

Confocal Laser Scanning Microscopy

Confocal microscopy of TUNEL-positive and control sperm was performedusing a BioRad MRC 1024 Laser Scanning Confocal Microscope equipped withan argon-ion laser (excitation at 488 nm and emission at 540 nm) andmounted on a Nikon Eclipse 600 series upright microscope. Confocalimages were obtained using a Nikon×100 (NA 1.4) numerical apertureobjective and Kalman collection filter. Digitized images were saved on aJaz disk (Iomega Corp., Roy, Utah) and processed with the AdobePhotoshop software (Adobe Systems, Mountain View, Calif.). Final imageswere printed using a Fuji Pictography 3000 (Fugi Photo Film Co., Tokyo,Japan) color printer.

Statistical Analysis

Results for the various numerical sperm functional parameters arepresented as mean±standard deviation (SD) values. Comparison betweenoxovanadium (IV) complex-treated and control sperm relative to spermmotility parameters and apoptosis were performed using paired,two-tailed Student's T-test. A p value of <0.05 was consideredsignificant. Non-linear regression analysis was used to find the EC₅₀values (i.e. concentrations of compound that result in 50% spermmotility loss) from the concentration effect curve using GraphPad Prismsoftware (San Diego, Calif.).

Results Oxovanadium (IV) Complexes of 1,10-Phenanthroline,2,2′-Bipyridyl and 5′-Bromo-2′-Hydroxyacetophenone and DerivativesDemonstrated Spermicidal

Activity

Oxovanadium (IV) complexes: phenanthroline (phen)-linked [VO(phen),VO(phen)₂,VO(Me₂—phen), VO(Me₂—phen)₂, VO(Cl—phen), and VO(Cl—phen)₂],bipyridyl (bipy)-linked [VO(bipy), VO(Me₂—bipy), and VO(Me₂—bipy)₂]viathe nitrogen-metal bond, and acetophenone (acph)-linked[VO(Br,OH—acph)₂]via oxygen-metal bond, were synthesized and tested themfor spermicidal activity using CASA. These complexes were testedside-by-side and at 8 different concentrations ranging from 1.9 μM to250 μM.

All of the tested oxovanadium complexes coordinated as ligands to thecentral vanadium (IV) ion induced concentration-dependent inhibition ofsperm motility assessed after a 3-hour incubation in BWW−0.3% BSAmedium. However, marked differences were noted in their potency. FIG. 2shows the concentration-response curves of spermicidal effects (loss ofsperm motility) of representative oxovanadium (IV) complexes, VO(phen),VO(phen)₂, VO(Cl—phen)₂, VO(Me₂—phen), VO(bipy)₂, VO(Me₂—bipy), andVO(Me₂—bipy)₂. Highly motile fractions of sperm were incubated for 3hours with increasing two-fold concentrations (1.9-250 μM) ofVO-complexes or DMSO alone in the assay medium. The percentage of motilesperm was evaluated by CASA. Each point represents the mean of from 2 to4 independent experiments.

Among the phenanthroline-linked cationic complexes,bis-1,10-phenanthroline complex, CO(phen)₂ and its 5-chloro derivative,VO(Cl—phen)₂, were the most potent, with EC₅₀ values of 6.5 μM and 5.5μM, respectively (see FIG. 2 and Table 1).

Table 1 shows comparative spermicidal activity analyzed by CASA andacrosomal loss analyzed by the flow cytometric anti-CD46 mAb bindingassay following exposure of sperm to oxovanadium(IV) complexescontaining either mono and bis 1,10-phenanthroline, 2,2′-bipyidyl or5′-bromo-2′-hydroxyacetophenone and derivatives.

TABLE 1 EC₅₀ Anti-CD46 positive Treatment (μM)^(ab) sperm (%)^(ac) DMSOcontrol NA 5.2 ± 0.7 VO(phen) 37 9.7 ± 0.7 VO(phen)₂ 6.5 9.9 ± 3.4VO(Me₂-phen) 20.5 14.0 ± 2.5^(d) VO(Me₂-phen)₂ 47.9 11.7 ± 1.1^(d)VO(Cl-phen) 39.1 11.2 ± 0.9 VO(Cl-phen)₂ 5.5 21.5 ± 0.5^(d) VO(bipy) 1186.1 ± 1.2 VO(bipy)₂ 35.3 4.7 ± 0.3 VO(Me₂-bipy) 83 6.4 ± 0.8VO(Me₂-bipy)₂ 72.5 3.2 ± 0.6 VO(Br,OH-acph)₂ 13.4 24.3 ± 0.6^(d) N-978.5 86 ± 2^(d) ^(a)Mean of two experiments. ^(b)Stock solutions (100mM) in DMSO were tested in serial 2-fold dilutions from 250 μM to 1.9μM. ^(c)Tested at 100 μM. ^(d)p < 0.05 compared with DMSO control.

The 5-bromo derivative of bis-2′-hydroxyacetophenone, a neutral complex,also demonstrated potent spermidical activity, with an EC₅₀ value of13.4 μM. Among the bipyridyl-linked cationic complexes, thebis-2,2′-bipyridyl complex, VO(bipy)₂, and its 4,7-dimethyl derivative,VO(Me₂—bipy)₂ were the most active, with EC₅₀ values of 35.3 μM and 72.5μM, respectively. The mono-2,2′-bipyridal complex, VO(bipy), was theleast active (EC₅₀=118 μM). Thus, the spermicidal activity ofoxovanadium (IV) complexes was strongly dependent on the coordinatedheteroligands.

Oxovanadium (IV) complexes having a bidentate ligand which formed a5-membered ring with the vanadium (IV) atom, a “butterfly structure,”demonstrated superior spermicidal activity when compared with theactivity of monodentate ligands (see FIG. 1, Table 2).

In Table 2, the apoptosis-inducing property of spermicidal oxovanadium(IV) complexes containing mono and bus 1,10-phenanthroline,2,2′-bipyridyl and 5′-bromo-2′-hydroxyacetophenone and derivatives isshown.

TABLE 2 JC-1 aggregate-positive Annexin V TUNEL sperm positive spermpositive sperm Treatment (%)^(a) (%)^(a) (%)^(a) DMSO control 92 ± 3 7 ±3 9 ± 1 VO(phen) 35 ± 12^(c) 97 ± 1^(c) 98 ± 1^(c) VO(phen)₂ 25 ± 1^(c)98 ± 1^(c) 98 ± 1^(c) VO(Me₂-phen) 38 ± 2^(c) 96 ± 3^(c) 97 ± 1^(c)VO(Me₂-phen)₂ 63 ± 2^(c) 17 ± 1 94 ± 1^(c) VO(Cl-phen) 39 ± 4^(c) 90 ±2^(c) 95 ± 1^(c) VO(Cl-phen)₂ 35 ± 1^(c) 99 ± 1^(c) 97 ± 1^(c) VO(bipy)95 ± 1 36 ± 8^(c) 43 ± 16^(c) VO(bipy)₂ 94 ± 1 26 ± 1 40 ± 22VO(Me₂-bipy) 88 ± 5 47 ± 16^(c) 45 ± 10^(c) VO(Me₂-bipy)₂ 62 ± 5^(c) 78± 7^(c) 63 ± 22^(c) VO(Br,OH-acph)₂ 83 ± 3 95 ± 1^(c) 98 ± 1^(c)^(a)Motile sperm were incubated at 37° C. for 3, 12, and 24 hoursrespectively, in either control medium, or in medium supplemented with100 μM each of the oxovanadium (IV) complexes, and stained respectivelywith either JC-1, FITC-Annexin V or with FITC-dUTP, and analyzed by flowcytometry. ^(b)Mean of two separate experiments. ^(c)p < 0.05 comparedwith DMSO control.

The concentrations of oxovanadium (IV) complexes with mono and bidentateancillary ligands, VO(phen), VO(phen)₂, VO(Me₂—phen), VO(Me₂—phen)₂,VO(Cl—phen), VO(Cl—phen)₂, VO(bipy), VO(bipy)₂, VO(Me₂—bipy),VO(Me₂—bipy)₂ and VO(Br, OH—acph)₂ that inhibited sperm motility by 50%(EC₅₀ values) calculated from concentration-response curve were 37 μM,6.5 μM, 20.5 μM, 47.9 μM, 39.1 μM, 5.5 μM, 118 μM, 35.3 μM, 83 μM, 72.5μM, and 13.4 μM, respectively (Table 1). These marked differences(21-fold) in potency of the spermicidal activity elicited by threeancillary heteroligands and their derivatives suggest that spermicidalpotency of oxovanadium (IV)-complexes is modulated by the coordinatedligands. The spermicidal activity of the most potent oxovanadium (IV)complex, VO(Cl—phen)₂, was 14-fold more potent than that of thecommercial detergent-based spermicide, N-9 (78.5 μM), when tested underidentical experimental conditions.

Also, in comparison to N-9, the spermicidal activity of oxovanadium (IV)complexes was not associated with a concomitant loss of acrosomalmembrane as quantitated by the flow cytometric anti-Cd46 mAb bindingassay using unfixed sperm suspension. Despite complete sperm motilityloss quantitated after a 3 hour incubation period, 76% to 97% of thetreated sperm remained anti-CD46 negative (acrosome-intact) (Table 1).The most potent oxovanadium (IV) complexes, VO(Cl—phen)₂ andVO(Br,OH—acph)₂ after a 3 hour incubation period induced a 4- to 5-fold(21.5% ±0.5% and 24.3% ±0.6% respectively, p<0.05) increase in acrosomereactions over control (5.2% ±0.7%), however, complete sperm motilityloss with these complexes was achieved within 2 and 10 minutes ofexposure. Thus, the spermicidal activity of oxovanadium (IV) complexeswas not concomitantly associated with disruption of sperm membranes.

Kinetics of Sperm Immobilization by Oxovanadium (IV) Complexes wasVariable

Interestingly, the kinetics of sperm immobilization by the oxovanadium(IV) complexes was variable. The corresponding times required for 50%motility loss of progressively motile sperm exposed to these complexesranged from <1 minute to >60 minutes. Sperm immobilization by theneutral complex, VO(Br,OH—acph)₂ was the fastest followed byVO(Cl—phen)₂ with T_(½) values of 38 seconds and 7.3 minutesrespectively. The other cationic oxovanadium (IV) complexes showed a lagperiod of 30-60 minutes to bring about >50% sperm motility loss. Bycomparison, sperm motility in control samples remained stable during the3-hour monitoring period.

Oxovanadium (IV) Complexes Affect Sperm Kinematics

The observed concentration- and time-dependent decrease in spermmotility after exposure to oxovanadium (IV) complexes were associatedwith significant changes in the centroid-derived movementcharacteristics of the surviving sperm, particularly with respect to thetrack speed (VCL), straight line velocity (VSL), and path velocity(VAP).

The representative sperm kinematic parameters observed for VO(Cl—phen)₂versus concentration and time are shown FIGS. 3A and 3B respectively.The effect of bis 5-chloro-1,10-phenanthroline oxovanadium (IV) sulfate,VO(Cl—phen)₂, on sperm motion parameters analyzed by CASA. In FIG. 3A,the concentration-dependent inhibition of sperm motility parameters isshown. Motile fractions of sperm were incubated in assay medium in thepresence of three increasing concentrations of VO(Cl—phen)₂ (0, 7.8,15.6, and 31.2 μM) for 3 hours at 37° C., and the centroid-derivedmotility characteristics were determined using the Hamilton-Thorne-IVOSversion 10 CASA. In FIG. 3B, the time-dependent effect of VO-complexeson sperm kinematics is shown. Motile fractions of sperm were incubatedfor 5, 10, and 15 minutes in assay medium in the presence of 200 μM ofVO(Cl—phen)₂, and the motility characteristics were determined by CASAas described under “Materials and Methods,” above. The sperm motionparameters were (left to right): MOT=motility (%); VCL=curvilinearvelocity (μm/s); VSL=straight line velocity (μm/s);l VAP=average pathvelocity (μm/s); STR=straightness, VSL/VAP (%); LIN=linearity, VSL/VCL(%); BCF=beat/cross frequency (Hz); and ALH=amplitude of lateral headdisplacement ((μm). Values are mean±SD of two representativeexperiments. Significant difference (P<0.05) between control andVO(Cl—phen)₂-treated sperm: progressive motility, VCL, VAP, and VSL.

The decreases in VCL, VSL, and VAP were similar in magnitude withincreasing concentrations of VO(Cl—phen)₂ or exposure time. However, thelinearity (LIN) of the sperm tracks and the straightness (STR) of theswimming pattern were affected only with increasing concentration of thedrug. The beat-cross frequency (BCF) and the amplitude of lateral spermhead displacement (ALH) were relatively uniform as the proportion ofmotile sperm declined with increasing concentration (0-15.6 μM) orexposure time (0-10 minutes). By contrast, the sperm motion parametersof control sperm showed insignificant changes during the 3-hourexposure.

Example 2 Oxovanadium (IV) Complexes of 1,10-Phenanthroline,1,2′-Bipyridyl, and 5′-Bromo-2′-Hydroxyacetophenone and DerivativesInduced Apoptosis

The oxovanadium (IV) complexes with phenanthroline, bipyridyl andacetophenone as ancillary ligands were analyzed for their ability toinduce apoptosis in human sperm. Three independent apoptosis assays wereused to quantitatively assess changes at the mitochondrial, surfacemembrane, and nuclear level. Analysis by flow cytometry of mitochondrialmembrane potential changes occurring during apoptosis were analyzed witha ΔΨm indicator, JC-1, a carbocyanine cationic dye by followingfluorescence associated with the uptake of JC-1 to evaluate ΔΨmmodifications as described in Smiley et al., 1991, (PNAS USA88:371-3675). Motile sperm exhibit intense green and red fluorescence ofJC-1, as shown in the flow cytometric quantitation, FIG. 4A.

Motile sperm were incubated at 37° C. in either control medium (0.1%DMSO) or medium supplemented with 100 μM of a representative oxovanadium(IV) complex, VO(Cl—phen)₂. The apoptosis-inducing ability ofVO(Cl—phen)₂ (FIGS. 4B, 4D, and 4F) in comparison with medium control(FIGS. 4A, 4C, and 4E) was tested by three flow cytometric assays thatquantitatively assess changes at the mitochondrial membrane potentialbased on JC-1 staining (FIGS. 4A and 4B); surface plasma membrane basedon FITC-Annexin V-staining (FIGS. 4C and 4D), and sperm nuclearcompartment based on FITC-dUTP nick-end labeling of fragmented DNA(FIGS. 4E and 4F) after 3, 12, and 24 hours respectively. Note themarked reduction in JC-1 red fluorescence (aggregates) labeling with noreduction in green emission (monomers). In FIGS. 4C-4F, sperm nucleiwere counter-stained with propidium iodide.

It can be seen that a 3 hour treatment with oxovanadium (IV) complex,VO(Cl—phen)₂, resulted in an extinction of the red fluorescence (FIG.4B) indicating that alteration occurs following VO(CL—phen)₂ treatment.A 3 hour pretreatment of sperm with 7 of the oxovanadium (IV) complexesresulted in variable decrease of ΔΨm-related fluorescence observed as31% to 73% reduction (p<0.05) in JC-1 aggregate (orange/green)fluorescence without concomitant reduction in JC-1 monomer (green)fluorescence (Table 2). By contrast, >90% of control sperm were positivefor orange/red fluorescence. The most potent spermicidal agents,VO(Cl—phen)₂ and VO(phen)₂ induced the maximum shift. Therefore, ΔΨmmodifications, evaluated by the uptake of cationic lipophilic dye, aredetected early in the process of apoptosis induced by oxovanadium (IV)complexes.

Changes in the plasma membrane of the cell surface also appear early incells undergoing apoptosis. In apoptotic cells, the membranephospholipid phosphatidyl serine is translocated from the inner to theouter leaflet of the plasma membrane, thereby exposing phosphatidylserine to the external cellular environment. Annexin V binds tophosphatidyl serine residues which are exposed on the surface of cellsundergoing apoptosis. The apoptosis-dependent surface binding ofFITC-labeled recombinant human Annexin V with 10 of the 11 testedoxovanadium (IV) complex-treated sperm showed a dramatic increase inbinding of Annexin V to sperm membrane (Table 2). After 12 hours ofincubation, 26% to 99% (p<0.05) of the treated sperm were apoptotic.Control sperm exhibited minimal fluorescence (FIG. 4C). By contrast, 99%of VO(Cl—phen)₂-treated sperm were positive for FITC-Annexin V (FIG. 4D)indicating that surface membrane alteration occurs following prolongedexposure to oxovanadium (IV) complexes. Control sperm treated with 0.1%of DMSO alone showed only 7±3% Annexin V positivity at 12 h. The mostpotent spermicidal complexes, VO(Cl—phen)₂, and VO(phen)₂, also inducedmaximum Annexin V positivity.

Next, TdT-mediated labeling of exposed 3′-OH termini for nuclear DNAwith FITC-conjugated dUTP by the in situ TUNEL method was employed todemonstrate that oxovanadium (IV) complexes induce apoptosis in thesperm nuclear compartment. FIGS. 4E and 4F depicts the two-colorcytometric contour plots of sperm nuclei of control sperm (E) treatedwith 0.1% DMSO, and test sperm (F) treated with 100 μM of VO(Cl—phen)₂in 0.1% DMSO after staining with FITC-dUTP and counterstaining with PI.Greater than 97% of VO(Cl—phen)₂ treated sperm became apoptotic(TUNEL-positive) after a 24 hour of incubation. A 24 hour exposure ofsperm with any one of the 11 oxovanadium (IV) complexes evaluatedresulted in marked increase of TUNEL-positive cells observed as 43% to98% (p<0.05) increase in FITC-dUTP fluorescence (Table 2). By contrast,<10% of control sperm treated with 0.1% DMSO alone showed apoptoticnuclei after a 24 hour of incubation. The percentages of apoptotic spermquantitated by the flow cytometric assays correlated well with thepotency (EC₅₀ values) of these oxovanadium (IV) complexes in spermimmobilization assays. Confocal images of TUNEL-positive sperm clearlyindicated that the fluorescence was localized to sperm nuclear region.

FIGS. 5A-5C show confocal microscopy images of sperm nuclei treated with100 μM VO(Cl—phen)₂ in 0.1% DMSO after incubation with TdT and FITC-dUTPwith (FIGS. 5A and 5C) and without (FIG. 5B) PI counterstaining. Nucleiof VO(CL—phen)₂-treated sperm showed dual fluorescence (FIG. 5C)consistent with apoptosis.

Confocal laser scanning microscopy images of sperm nuclei undergoingapoptosis following treatment with a oxovanadium (IV) complex,VO(Cl—phen)₂. Motile sperm were incubated for 24 hours in mediumsupplemented with 100 μM VO(Cl—phen)₂, fixed, permeabilized, andvisualized for DNA degradation in a TUNEL assay using TdT and FITC-dUTP.FIG. 5A shows sperm nuclei counterstained with PI (red color). FIG. 5shows sperm nuclei visualized for dUTP incorporation using FITC-dUTP(green). FIG. 5C shows nuclei of VO(Cl—phen)₂-treated sperm visualizedfor dual fluorescence. Apoptotic nuclei appear yellow due tosuperimposed labels. Original magnification ×1000.

DISCUSSION

The above described results provide unprecedented evidence thatoxovanadium (IV) complexes with 1,10-phenanthroline, 2,2′-bipyridyl, or5′-bromo-2′-hydroxyacetophenone and their derivatives linked to vanadiumvia nitrogen or oxygen bond have potent spermicidal activity againsthuman sperm. The order of spermicidal efficacy for oxovanadium (IV)complexes synthesized and evaluated was:VO(Cl-phen)₂>VO(phen)₂>VO(Br,OH-acph)₂>VO(Me₂-phen)>VO(bipy)₂>VO(phen)>VO(Cl-phen)>VO(Me₂-phen)₂>VO(Me₂-bipy)₂>VO(Me₂-bipy).Thus, despite the structural similarities of phenanthroline andbipyridyl rings, the phenanthroline complexes of oxovanadium (IV),particularly the bis-phenanthroline complex, VO(Cl-phen)₂, were the mostactive and the mono bipyridal complex, VO(bipy), being the least active.In general, the oxovanadium (IV) complexes stabilized by bidentateligands which formed a 5-membered ring with vanadium (IV) atom were 3-to 7-fold more potent when compared with monodentate complexes.

The kinetics of sperm immobilization by these oxovanadium (IV) complexesattached to various ancillary ligands was dependent on their net charge.Comparative structure-activity relationship analyses of vanadocenes(U.S. Ser. No. 09/008,989) and the oxovanadium (IV) complexes of theinvention clearly demonstrated that the spermicidal properties of theorganovanadium (IV) complexes and oxovanadium (IV) complexes wasdependent upon the central vanadium (IV) ion within these complexes, thevarious ancillary ligands linked by carbon, nitrogen, or oxygen bonds tocentral vanadium (IV) ion significantly contributed either to finetuning of the spermicidal potency or enhancing the stability of thesecomplexes in aqueous solution. In addition, similar to our earlierfindings with neutral complexes of vanadocenes, the neutral complex ofoxovanadium (IV), VO(Br,OH-acph)₂ rapidly inactivated sperm incomparison to the cationic oxovanadium (IV) complexes or cationicvanadocenes which required a lag period of several minutes.

Therefore, it appears from our study that despite the tetrahedralgeometry of the “bent-sandwich” structures of vanadocenes and the squarepyramidal geometry or the “butterfly” structures of oxovanadium (IV)complexes, the rapidity of vanadium (IV)-dependent spermicidal activitywas dependent on neutral charge of these complexes. The fact that theneutral complex of oxovanadium (IV), VO(Br,OH-acph)₂ induced both apotent and rapid sperm motility loss suggests that this complex ofoxovanadium (IV) is rapidly transported across the sperm cell membranes.Because of its rapidity (sperm immobilization T_(1/2)=38 seconds) andpotency, VO(Br,OH-acph)₂ provides a useful contraceptive agent.

The mechanism of sperm motility loss induced by oxovanadium (IV)complexes is unknown. Both the vanadocenes and oxovanadium (IV)complexes of vanadium (IV) have antitumor activity (Sakurai et al.,1995, BBRC, 206:133-137). The antitumor effects of vanadium (IV)complexes are thought to be due to their reaction with H₂O₂ forminghydroxyl radical in a Fenton-like reaction. In particular, theoxovanadium (IV) complex, following dissociation of phenanthroline ringsleads to the formation of peroxocompounds which in the presence of H₂O₂generates hydroxyl radicals. In support of this hypothesis is theobservation that vanadyl-phen complex induces hydroxyl radical-dependentDNA cleavage in the presence of H₂O₂. The vanadyl complex,[VO(phen)(H₂O₂)₂]₂ ⁺ has high antitumor activity toward humannasopharyngeal carcinoma. Hydrogen peroxide is formed in cells bydismutation of superoxide anions which are generated in various systemssuch as xanthine-oxidase, NADPH oxidase and NADH-dependent cytochromeP-450 and neutrophils. Thus, H₂O₂ is thought to react with oxovanadium(IV)-bound to DNA to generate ROS resulting in cleavage of DNA. It islikely that oxovanadium (IV)-induced sperm motility loss and apoptosisare mediated primarily by the ability to these complexes to induceROS-mediated damage to sperm. In sperm, an NADPH-dependent superoxidegenerating system has been demonstrated (Kessopoulou et al, 1992, J.Reprod. Fert 94:463-470). In addition, the ability of H₂O₂ generatingLactobacillus acidophilus which is present in the vaginas of most normalwomen has the ability to further potentiate the spermicidal activity ofintravaginally applied oxovanadium (IV) complex. This is in contrast tothe commercial vaginal detergent spermicide, N-9, which is selectivelytoxic to Lactobacilli. Furthermore, unlike N-9, the spermicidal activityof the inorganic coordination complexes of vanadium (IV) was notconcomitantly associated with membrane disruption.

The irreversible nature of the spermicidal activity of oxovanadium (IV)complexes is likely due to their ability to induce apoptosis. Threeindependent methods were used to quantitatively assess apoptotic changesin the mitochondria, surface membrane, and nuclear compartment.Mitochondria are the primary targets for apoptosis and alterations inmitochondrial structure and function are early events of apoptotic celldeath. These studies demonstrated that spermicidal oxovanadium (IV)complexes induced depolarization of sperm mitochondria, an early markerfor apoptotic cell death. Prolonged exposure of sperm to thesespermicidal complexes also resulted in increased FITC-Annexin V bindingto sperm surface due to membrane changes during apoptosis, as well asincreased dUTP incorporation in the nuclei of treated sperm. Sincevanadium (IV) compounds by themselves do not cleave DNA, the dramaticuptake of dUTP incorporation observed in our study appears to be due tothe cleavage of the DNA polymer by the cytotoxic effects of reactiveoxygen species-mediated effects of oxovanadium (IV) complexes. The factthat human sperm are exquisitely sensitive to oxidative stress, and theability of vanadium (IV)-containing inorganic coordination complexes topotentiate these effects would make these oxovanadium (IV) complexes asa new class of gentle contraceptive agents.

The specification includes many references to patents and publishedliterature, each of which is hereby incorporated by reference, for allpurposes, as if fully set out herein.

We claim:
 1. A method of inhibiting motility of sperm comprisingcontacting sperm with an organometallic oxovanadium IV complexcomprising at least one substituted or unsubstituted ligand that is a1,10 phenanthroline; a bridged or unbridged 2,2′ bipyridyl; a2′-hydroxyacetophenone; or a 3-hydroxy-propenal.
 2. The method of claim1, wherein said complex comprises oxovanadium (IV) complexed with atleast one bidentate ligand.
 3. The method of claim 1, wherein saidcomplex has the formula:

where R⁴, R⁵ and R⁶ are the same or different and are eachindependently: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl,cyano, carboalkoxy or nitro; X⁴ and X⁵ are the same or different and areeach independently: monodentate or bidentate ligands; and n is 0 or 1.4. The method of claim 1, wherein said complex has the formula:

wherein R, and R¹ are the same or different and are each independently:H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano,carboalkoxy or nitro; X and X¹ are the same or different and are eachindependently: monodentate or bidentate ligands; and n is 0 or
 1. 5. Themethod of claim 1, wherein said complex has the formula:

where R⁷ and R⁹ are the same or different and are each independently: H,lower alkyl, lower alkoxy, or halogenated alkyl; R⁸ is H, lower alkyl,halo, lower alkoxy, or halogenated alkyl; Y and Y¹ are the same ordifferent and are each independently: monodentate or bidentate ligands;and n is 0 or
 1. 6. The method of claim 2, wherein the bidentate ligandcomprises an N, N′ bidentate ligand.
 7. The method of claim 6 whereinthe N, N′ bidentate ligand comprises a bipyridyl.
 8. The method of claim7, wherein the bipyridyl comprises a 2, 2′ bipyridyl.
 9. The method ofclaim 8, wherein the oxovanadium IV complex is selected from the groupconsisting of: (diaqua)(2,2′-bipyridyl)oxovanadium(IV) sulfate;(aqua)bis(2,2′-bipyridyl)oxovanadium(IV) sulfate;(diaqua)(4,4′-dimethyl-2,2′-bipyridyl)oxovanadium(IV) sulfate; and(aqua)bis(4,4′-dimethyl-2,2′-bipyridyl)oxovanadium(IV) sulfate.
 10. Themethod of claim 6, wherein the N, N′ bidentate ligand comprises abridged bipyridyl.
 11. The method of claim 10, wherein the bridgedbipyridyl comprises a phenanthroline.
 12. The method of claim 11,wherein the oxovanadium IV complex is selected from the group consistingof: (diaqua)(1,10-phenanthroline)oxovanadium(IV) sulfate;(aqua)bis(1,10-phenanthroline)oxovanadium(IV) sulfate;(diaqua)(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate;(aqua)bis(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate;(diaqua)(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate; and(aqua)bis(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate.
 13. Themethod of claim 6 wherein the N, N′ bidentate ligand comprises adiamine.
 14. The method of claim 13, wherein the diamine comprises oneof phenanthroline and bipyridyl.
 15. The method of claim 2, wherein thebidentate ligand comprises and N, O bidentate ligand.
 16. The method ofclaim 15, wherein the N,O bidentate ligand comprises an amino acidfunctional group.
 17. The method of claim 15, wherein the N,O bidentateligand comprises Schiff base functional group.
 18. The method of claim2, wherein the bidentate ligand comprises an O, O′ bidentate ligand. 19.The method of claim 18, wherein the an O, O′ bidentate ligand comprisesa dicarboxylate compound.
 20. The method of claim 18, wherein the an O,O′ bidentate ligand comprises a 2-hydroxyacetophenone compound.
 21. Themethod of claim 20, wherein the oxovanadium IV complex comprisesbis(5′-bromo-2′-hydroxyacetophenone) oxovanadium (IV).
 22. The method ofclaim 18, wherein the an O, O′ bidentate ligand comprises aacetylacetone compound.
 23. The method of claim 18, wherein the an O, O′bidentate ligand comprises a catechol compound.
 24. The method of claim3, wherein X⁴ and X⁵ comprise monodentate ligands.
 25. The method ofclaim 24, wherein the monodentate ligands are each independently H₂O,halide, or carboxylate.
 26. The method of claim 3, wherein X⁴ and X⁵together form a bidentate ligand.
 27. The method of claim 26, whereinthe bidentate ligand is an N, N′ bidentate ligand.
 28. The method ofclaim 27 wherein the N, N′ bidentate ligand is a bipyridyl or a bridgedbipyridyl.
 29. The method of claim 28 wherein the N, N′ bidentate ligandcomprises a bridged bipyridyl, wherein the bridged bipyridyl comprises aphenanthroline.
 30. The method of claim 27 wherein the N, N′ bidentateligand comprises a diamine.
 31. The method of claim 26, wherein thebidentate ligand comprises an N, O bidentate ligand.
 32. The method ofclaim 31, wherein the N,O bidentate ligand is a ligand comprising anamino acid functional group or a ligand comprising a Schiff basefunctional group.
 33. The method of claim 26, wherein the bidentateligand comprises an O, O′ bidentate ligand.
 34. The method of claim 33,wherein the O, O′ bidentate ligand is a dicarboxylate compound, a2-hydroxy acetophenone compound, an acetylacetone compound, or acatechol compound.
 35. The method of claim 1, wherein the complexcomprises oxovanadium (IV) complexed with at least one of a 1,10phenanthroline, a 2,2′-bipyridyl, and a 2-hydroxyacetophenone.
 36. Themethod of claim 35, wherein the oxovanadium complex comprisesoxovanadium (IV) complexed with at least one monodentate ligand.
 37. Themethod of claim 36, wherein the monodentate ligand is H₂O, halide, orcarboxylate.
 38. The method of claim 4, wherein X and X¹ comprisemonodentate ligands.
 39. The method of claim 38, wherein the monodentateligands are each independently H₂O, halide, or carboxylate.
 40. Themethod of claim 4, wherein X and X¹ together form a bidentate ligand.41. The method of claim 40, wherein the bidentate ligand comprises an N,N′ bidentate ligand.
 42. The method of claim 41 wherein the N, N′bidentate ligand is a bipyridyl or a bridged bipyridyl.
 43. The methodof claim 42 wherein the N, N′ bidentate ligand comprises a bridgedbipyridyl, wherein the bridged bipyridyl is phenanthroline.
 44. Themethod of claim 41 wherein the N, N′ bidentate ligand comprises adiamine.
 45. The method of claim 40, wherein the bidentate ligandcomprises an N, O bidentate ligand.
 46. The method of claim 45, whereinthe N,O bidentate ligand is a ligand comprising an amino acid functionalgroup or a ligand comprising a Schiff base functional group.
 47. Themethod of claim 40, wherein the bidentate ligand comprises an O, O′bidentate ligand.
 48. The method of claim 47, wherein the O, O′bidentate ligand is a dicarboxylate compound, a 2-hydroxyacetophenonecompound, an acetylacetone compound, or a catechol compound.
 49. Themethod of claim 5, wherein Y and Y¹ comprise monodentate ligands. 50.The method of claim 49, wherein the monodentate ligands are eachindependently H₂O, halide, or carboxylate.
 51. The method of claim 5,wherein Y and Y¹ together form a bidentate ligand.
 52. The method ofclaim 51, wherein the bidentate ligand comprises an N, N′ bidentateligand.
 53. The method of claim 52 wherein the N, N′ bidentate ligand isbipyridyl or bridged bipyridyl.
 54. The method of claim 53 wherein theN, N′ bidentate ligand comprises a bridged bipyridyl, wherein thebridged bipyridyl is phenanthroline.
 55. The method of claim 52 whereinthe N, N′ bidentate ligand comprises a diamine.
 56. The method of claim51, wherein the bidentate ligand comprises an N, O bidentate ligand. 57.The method of claim 56, wherein the N,O bidentate ligand is a ligandcomprising an amino acid functional group or a ligand comprising aSchiff base functional group.
 58. The method of claim 51, wherein thebidentate ligand comprises an O, O′ bidentate ligand.
 59. The method ofclaim 58, wherein the O, O′ bidentate ligand is a dicarboxylatecompound, a 2-hydroxyacetophenone compound, an acetylacetone compound,or a catechol compound.
 60. The method of claim 1, wherein theoxovanadium (IV) complex comprises: bis(5′-bromo-2′-hydroxyacetophenone)oxovanadium(IV); or(aqua)bis(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate.
 61. Themethod of claim 1, wherein the pharmaceutical vehicle is an intravaginalinsert.
 62. The method of claim 1, wherein the oxovanadium IV complexcomprises a complex including a bridged bipyridyl, the complex havingthe formula:

wherein R² and R³ are the same or different and are each independentlyH, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano,carboalkoxy or nitro; X² and X³, are the same or different and are eachindependently monodentate or bidentate ligands; Z is O, CH₂, CH₂—CH₂, orCH═CH; and n is 0 or
 1. 63. The method of claim 62, wherein X² and X³comprise monodentate ligands and are each independently H₂O, halide, orcarboxylate.
 64. The method of claim 62, wherein X² and X³ together forma bidentate ligand.
 65. The method of claim 64, wherein the bidentateligand comprises an N, N′ bidentate ligand.
 66. The method of claim 64,wherein the bidentate ligand comprises an N, O bidentate ligand.
 67. Themethod of claim 64, wherein the bidentate ligand comprises an O, O′bidentate ligand.
 68. A method of inhibiting of sperm comprisingcontacting sperm with an a composition comprising a spermicidaleffective amount of an organometallic oxovanadium (IV) complex includingat least one bidentate ligand comprising a structure selected from thegroup consisting of a diamine, an amino acid functional group, a Schiffbase functional group, a dicarboxylate, a 2-hydroxyacetophenone, anacetylacetone group, and a catechol group.
 69. A method of inhibitingmotility of sperm comprising contacting sperm with a compositioncomprising a spermicidal effective amount of an organometallicoxovanadium (IV) complex comprising at least one substituted orunsubstituted ligand that is a 1,10 phenanthroline; a bridged orunbridged 2,2′ bipyridyl; a 2′-hydroxyacetophenone; or a3-hydroxy-propenal, and a pharmaceutically acceptable carrier, diluentor vehicle.
 70. A method of inhibiting motility of sperm comprisingcontacting sperm with an a composition comprising a spermicidaleffective amount of an organometallic oxovanadium (IV) complex selectedfrom the group consisting of: (diaqua)(2,2′-bipyridyl)oxovanadium(IV)sulfate; (aqua)bis(2,2′-bipyridyl)oxovanadium(IV) sulfate;(diaqua)(4,4′-dimethyl-2,2′-bipyridyl)oxovanadium(IV) sulfate;(aqua)bis(4,4′-dimethyl-2,2′-bipyridyl)oxovanadium(IV) sulfate;(diaqua)(1,10-phenanthroline)oxovanadium(IV) sulfate;(aqua)bis(1,10-phenanthroline)oxovanadium(IV) sulfate;(diaqua)(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate;(aqua)bis(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate;(diaqua)(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate;(aqua)bis(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate; andbis(5′-bromo-2′-hydroxyacetophenone) oxovanadium(IV).